Allergen bronchoprovocation: correlation between FEV1 maximal percent fall and area under the FEV1 curve and impact of allergen on recovery

Background House dust mite (HDM) induces greater responses than other allergens during allergen bronchoprovocation (ABP) testing. The two standardized methods for reporting results of ABP tests are the maximal percent fall in forced expiratory volume in one second (FEV1, max; %) and the area under the FEV1 vs time curve (AUC; %FEV1 x min). The relationship between these methods has not been previously investigated. Aims We aimed to measure the correlation between FEV1, max and AUC during the early asthmatic response (EAR) and the late asthmatic response (LAR), and to determine if the EAR recovery period for HDM would be longer than other allergens (cat, grass, horse, and ragweed). Methods We retrospectively calculated the AUC and correlation between FEV1, max and AUC during the EAR(0-2 h) and LAR(3-7 h) for each allergen. We compared EAR(0-3 h) and LAR(3-7 h) FEV1, max, AUC and absolute difference in FEV1, max to the most recovered FEV1 (FEV1, min). We performed pairwise comparisons of correlation and slope values using Fischer’s r to z transformation and t-tests, respectively. AUC and absolute differences in FEV1, max and FEV1, min were compared using a one-way ANOVA test, followed by a post-hoc Scheffe test. Results Correlation between the FEV1, max and AUC during the EAR(0-2 h) (n = 221) was 0.807, and was 0.798 during the LAR(3-7 h) (n = 157 of 221), (difference p = 0.408). The EAR(0-3 h) AUC and FEV1, max did differ between allergens (both p < 0.0001) but the LAR(3-7 h) AUC and FEV1, max did not (p = 0.548 and 0.824, respectively). HDM did not have a larger AUC or FEV1, max, than all other allergens during the EAR(0-3 h) or the LAR(3-7 h). The absolute difference between the FEV1, max and FEV1, min during the EAR(0-3 h) did not differ between allergens (p = 0.180). Conclusion The FEV1, max and AUC for both the EAR(0-2 h) and LAR(3-7 h) had excellent correlation, with no significant difference. Thus, significant bronchoconstriction will likely result in a longer recovery period. There was no evidence of delayed EAR(0-3 h) recovery following HDM challenges, so HDM did not induce a larger response compared to all the other allergens examined. Registration: Not registered. This is not a clinical trial. (This study is a retrospective analysis of data collected during several registered trials.)


Introduction
The allergen bronchoprovocation (ABP) test is used to study asthma pathophysiology and pharmacological agents and is performed by administering serial concentrations of a relevant allergen to induce bronchoconstriction. The early asthmatic response (EAR) is defined as a ≥ 20% decrease in forced expiratory volume in one second (FEV 1 ) and will usually resolve without treatment within 2 h [1,2]. Bronchoconstriction during the EAR is usually greatest within 20 min postchallenge [3]. Late asthmatic responses (LARs), which are characterized by bronchoconstriction defined as a ≥ 15% decrease in FEV 1 during the 3 to 7 h (or longer) timeframe post-challenge are reported to manifest in as few as 34.5% [3], and as many as 50% of early responders which may depend on geographical location [1,2].
There are two units of measure used to quantify ABP results: the maximal percent fall in FEV 1 (FEV 1, max ) and the area under the FEV 1 versus time curve (AUC). While the FEV 1, max provides insight on the severity of bronchoconstriction, the AUC includes the duration of the response. Generally, AUC is preferred over FEV 1, max because it is less sensitive to outliers [2]. Nonetheless, both methods are considered reproducible and sensitive [4,5]. A relationship must exist between the FEV 1, max and AUC values, but their correlation has not been previously investigated. Understanding the relationship of the two units of measure will help guide future research when deciding which, if not both, measure can be used. Pharmacological studies utilizing ABP tests are often most interested in the recovery, and the FEV 1, max and AUC are both used to represent recovery in different ways. The correlation between FEV 1, max and AUC would indicate how well these two interpretations of recovery are related: whether the degree of bronchoconstriction reflects the duration of the response. Calculating both the FEV 1, max and AUC would be valuable if the values were not well linked. However, if the valued had a strong association, this would mean one value could help predict the other. In addition, the longer time period of the LAR could allow for a greater variation in AUC since it is dependent on the length of the response, while the FEV 1, max only depends on the time point with the greatest degree of bronchoconstriction. Thus, a strong correlation between the two measures would also show that the AUC is a useful measure even during longer recovery periods like the LAR.
In addition, the choice of allergen administered during the ABP test can impact the response. House dust mite (HDM) allergen has been shown to cause greater ABP responses. Previous research has found that HDM caused a larger FEV 1, max during the LAR when compared to pollen challenges with EARs of similar magnitude [6]. Furthermore, HDM caused greater FEV 1, max at every time point compared to cat allergen [7]. All allergens activate the immunoglobulin-E (IgE) pathway to cause mast cell and basophil degranulation resulting in bronchoconstriction [8]. However, HDM may cause more severe bronchoconstriction by activating additional proteolytic pathways [9]. A major factor in bringing about an immune response to HDM allergen may be Der p 1, which has shown cysteine protease activity; it was found to cleave proteins on the IL-2 receptor [10,11], leading to a possible immune bias for T H 2 cells, increasing allergic hypersensitivity [9]. Chronic exposure to HDM may also increase responsiveness to an ABP challenge, contributing to greater responses. [6]. Determining if HDM is associated with a longer recovery period after the EAR would provide further evidence that this allergen causes more severe outcomes in ABP tests and help support the current understanding of the excessive bronchoconstriction compared to other allergens. The existing research on the response to HDM allergen is focused on the FEV 1, max ; we expanded on this understanding by also measuring the AUC. We also included a larger number of both seasonal and perennial allergens (cat, grass, HDM, horse, and ragweed) to compare the responses to a greater variety of allergens. The focus on recovery after an HDM challenge is also clinically relevant, as it will help put the theoretical understanding of the additional proteolytic pathway HDM may activate, in terms of measures of recovery.
One of our objectives was to understand how strong the relationship is between FEV 1, max and AUC during the EAR (0-2 h) as well as during the LAR (3-7 h) . We hypothesized that the FEV 1, max and AUC should have good to excellent correlation (r ≥ 0.8) for both the EAR (0-2 h) and the LAR (3-7 h) ; but, we suspected it would be greater for the EAR (0-2 h) based on the nature of the ABP test, with allergen administration stopping once a 20% fall in FEV 1 is reached. Understanding the correlation between these endpoints is important to ABP tests: it will allow us to determine how the magnitude or duration of the response is linked to FEV 1, max . We also set out to determine if the EAR recovery after an HDM challenge would be significantly longer than that of other allergens.

Methods
We retrospectively gathered ABP data from our Clinical Investigator Collaborative database using data from the University of Saskatchewan and McMaster University. Participants included non-smokers aged 18 to 63 who took part in studies conducted from 2002 to 2019. We looked at screening allergen challenges which do not include any treatment intervention. The allergens examined, namely, cat, grass, HDM, horse, and ragweed, were selected based on the study participant's clinical history and allergen prick skin test results [1,2]. Participants who took part in more than one study had their data included only once, using the most recent data. ABP tests were performed as previously described [1,2]. Participants inhaled allergens selected based on their skin prick test results in conjunction with their own reporting of allergy symptoms. FEV 1 was measured 10 min, 20 min, 30 min, 45 min, 60 min, 90 min, 2 h, 3 h, 4 h, 5 h, 6 h, and 7 h post-challenge. The EAR (0-2 h) FEV 1, max was the largest percent fall in FEV 1 relative to baseline (i.e., pre-allergen inhalation) from 0 to 2 h post-challenge, and the LAR (3-7 h) FEV 1, max was the largest percent fall in FEV 1 from 3 to 7 h post-challenge; each being at least 20% and 15% respectively. If a single time point during the ABP test was missing, it was estimated using a weighted average of the preceding and following percent fall in FEV 1 values. AUC for the EAR (0-2 h) and LAR (3-7 h) were calculated from percent fall in FEV 1 versus time data using the trapezoid rule. The absolute difference in FEV 1, max and the most recovered (i.e., least) percent fall in FEV 1 (FEV 1, min ) that followed during EAR (0-3 h) was calculated to determine the magnitude of recovery.
Scatterplot graphical representations for correlation analyses were constructed for all allergens combined and for individual allergens (cat, grass, HDM, horse, and ragweed) both for the EAR (0-2 h) and LAR (3-7 h) . Pearson's correlation coefficient and the slope of the regression line were calculated using Microsoft Excel (Version 16.60). Pairwise comparisons of correlation were done using Fischer's r to z transformation [12]. Pairwise comparisons of slopes were done using a t-test [12]. AUC and absolute differences in FEV 1, max and FEV 1, min were compared using a one-way ANOVA test. A significant ANOVA test was followed by a posthoc Scheffe test. Significance at the 5% level was tested.
We believed that a slower recovery period after an HDM challenge would manifest as a larger EAR (0-3 h) AUC; the third hour was included to ensure we were measuring recovery at the last stage of the EAR, allowing us to see if any participants still had significant bronchoconstriction. Determining if HDM is associated with a longer recovery period after the EAR would provide further evidence that this allergen causes more severe outcomes in ABP tests and help support the current understanding of the excessive bronchoconstriction compared to other allergens.

Participant characteristics
Data from 221 participants were used for EAR (0-2 h) analysis, 157 of the 221 participants (71%) were dual responders, defined as a ≥ 15% decrease in FEV 1 during the 3 to 7 h post-challenge, and these data were used for LAR (3-7 h) analyses. Three participants had a single time point missing during the ABP test (one each at 20 min, 30 min, and 45 min). The most common allergen used for ABP testing was HDM followed by cat, grass, ragweed, and horse ( Table 1).

Overview of EAR and LAR responses
Mean fall in FEV 1 at each timepoint post-inhalation for each allergen are shown in Fig. 1. During the whole 7-h post-challenge timeframe, ragweed inhalation generated the largest fall in FEV 1 (36.9%) followed by HDM (35.5%), grass (31.5%), cat (30%) and horse (29.7%). Recovery from ragweed inhalation required the most time (i.e., largest EAR (0-3 h) AUC) and is the least complete (8.8% bronchoconstriction remains at 3 h post-challenge).
Cat and horse exhibit the least bronchoconstriction and recover more quickly (i.e., small EAR (0-3 h) AUCs) and more completely (i.e., FEV 1 returns to within 5% of baseline at 3 h post-challenge). LAR responses are relatively similar across the 3-7-h post-challenge timeframe in terms of both maximal fall in FEV 1 and AUC for cat, horse and HDM; these responses are developing gradually hour by hour. Late responses to grass develop rapidly between 4 and 6 h and beginning to recover at 7 h. The FEV 1 decrease during the LAR to ragweed is initially slight (approximately 3% over hours 4 and 5) then nearly doubles over the next two hours with a steep downward trend and no evidence of recovery at 7 h post-challenge.

Discussion
The correlation of FEV 1, max to AUC for all allergens combined during the EAR (0-2 h) and LAR (3-7 h) were both strong and did not differ statistically (r = 0.807 and 0.798 respectively; difference p = 0.408). Thus, a large FEV 1, max correlates to a large AUC for both the EAR (0-2 h) and LAR (3-7 h) . This result is useful for future ABP tests, since it establishes a strong relationship between FEV 1, max and AUC for both the EAR (0-2 h) and Thus, a greater degree of bronchoconstriction, as measured by the FEV 1, max , will likely result in a longer recovery period, as seen by the AUC. Future pharmacological studies aimed at measuring the recovery after an allergen-induced asthmatic response might pick either measure to demonstrate recovery, or choose a specific measurement based on the correlations outlined for the chosen allergen. The LAR (3-7 h) occurs over a longer time period than the EAR (0-2 h) , which we believed would allow for greater variability in AUC, thereby reducing the correlation between FEV 1, max and AUC; since these two correlation values did not differ, the AUC method can provide insight on the magnitude of response even during longer response periods. A previous study also found that the correlation between FEV 1 and the area under the expiratory flow-volume curve in a methacholine challenge was strong (r = 0.939) [13]. Some key differences from that study are that the expiratory flow-volume curve and AUC are not direct substitutes for each other, and methacholine and ABP challenges do not cause bronchoconstriction through the same pathway with substantially different time courses; methacholine-induced bronchoconstriction is more rapid in both onset and recovery. Methacholine is a direct bronchoconstrictor, it binds to receptors on airway smooth muscle, while an ABP challenge is an indirect test that leads to bronchoconstriction through inflammatory mediators via the IgE pathway Pairwise comparisons of individual allergens' correlation of FEV 1, max vs AUC showed grass (r = 0.935) had a statistically significant higher value compared to cat, HDM, horse, and ragweed during the EAR (0-2 h) (p values range from < 0.0001 to 0.042). The betweenparticipant variability of FEV 1, max and AUC for grass allergen tended to be less than that for other allergens. The allergen with the second highest correlation during the EAR (0-2 h) was ragweed (r = 0.839), although this value only differed statistically to cat and grass (p = 0.035 and 0.042, respectively). Both grass and ragweed are seasonal allergens. ABP testing in these individuals was performed outside allergy season to avoid the potential for increased allergen responsiveness resulting from recent exposure. This is in contrast to perennial allergens like HDM, wherein exposure is chronic, potentially leading to enhanced responsiveness to ABP testing [6]. HDM had a lower correlation coefficient (r = 0.778) but only differed statistically to grass (p = 0.001). Perhaps the difference in correlation is due to the type of exposure: chronic exposure to an allergen leading to more responsive airways maybe associated with more between-participant variability, leading to a lower AUC vs FEV 1, max correlation. Nonetheless, cat allergen, had the lowest correlation (r = 0.650), and would only be a   (3-7 h) was steeper than that of the EAR (0-2 h) (slope = 161.1 and 81.9 respectively; p < 0.0001). During the EAR (0-2 h) , the only significant pairwise comparisons for the slope of FEV 1, max vs AUC was cat vs grass (slope = 70.6 and 102.9 respectively; p = 0.013) and grass vs HDM (slope = 102.9 and 71.7 respectively; p = 0.002). The EAR (0-2 h) slope is related to the recovery period following allergen inhalation (once FEV 1, max is reached, FEV 1 would approach baseline and AUC would decrease), whereas the LAR (3-7 h) slope is a function of the magnitude of the LAR (3-7 h) (i.e., the development of the response as a sustained drop in FEV 1 which would result in a large AUC). No one allergen resulted in a difference in recovery after allergen inhalation (i.e., EAR (0-2 h) ), or in the magnitude of the LAR (3-7 h) , than all other allergens. Specifically, since HDM did not have significantly different slopes, we cannot conclude that HDM caused a longer recovery period, or a larger LAR (3-7 h) magnitude.
The slope values may also be influenced by outliers, especially at larger FEV 1, max values (≥ 45) where the points were much more dispersed.
Based on Fig. 1, it is possible that grass is the only allergen undergoing recovery during the 6-to-7-h period during the LAR (3-7 h) . All other allergens appear to still be increasing or reaching their maximum FEV 1 at 7 h, but we would need to have data past this time point, until the maximum response is reached, to be able to comment on LAR (3-7 h) recovery. During the EAR (0-3 h) , ragweed had both the largest FEV 1, max and AUC, followed by HDM. However, these values are not significantly different compared to the other allergens. During the LAR (3-7 h) the allergen with the largest FEV 1, max and AUC were not the same: HDM had the largest FEV 1, max while grass had the largest AUC. Importantly though, neither the LAR (3-7 h) FEV 1, max nor the AUC values differed statistically between allergens.
The absolute difference in the highest and lowest percent fall in FEV 1 during the EAR (0-3 h) did not differ between allergens (p = 0.180), while the EAR (0-3 h) AUC did (p < 0.0001). However, HDM did not result in a larger AUC than all the other allergens; the only significantly different pairwise comparisons were cat vs HDM, cat vs ragweed, and horse vs ragweed. No single allergen had a statistically larger EAR (0-3 h) AUC than the rest. Thus, we cannot conclude from these findings that the recovery after the EAR (0-3 h) for HDM allergen is longer than other allergens, the same conclusion we reached when comparing EAR (0-2 h) FEV 1, max vs AUC slopes. We suspected that HDM would result in a delayed or slower recovery during the EAR (0-3 h) because of previous research showing more severe ABP results [6,7], as well as the activation of additional proteolytic pathways that other allergens may not induce [9][10][11]. Our findings may not be in accordance with previous data due to ragweed having the largest EAR (0-3 h) AUC, Table 3 Mean FEV 1 and AUC (± SD) data and statistical analyses for common allergens